Self-renewal and differentiation by spermatogonial stem cells (SSCs) provides the foundation for continual spermatogenesis. Thus, sustained fertility in males and preservation of genetic lines is critically dependent on SSC biological function. Spermatogenesis is a classic stem cell-dependent process in which SSC function is controlled by external niche stimuli and internal gene expression. Currently, mechanisms regulating these processes in SSCs are poorly understood. Because there are currently no known specific morphological or molecular markers for SSCs, they cannot be isolated and are defined solely by function. Therefore, it is extremely challenging to study the internal molecular mechanisms regulating SSC biological function in vivo. Recently, a chemically defined culture system has been developed that supports a germ cell population enriched for self-renewing SSCs for extended periods of time. Utilizing this culture system it is now possible to define the genes that are essential for SSC functions. Moreover, when utilized in conjunction with functional transplantation, direct effects of specific genes on SSC activity in vitro can be unequivocally determined. RNA interference (RNAi) is a powerful tool for deciphering molecular pathways and assessing the biological significance of specific genes. Recent technological advances allow for generation of stable and inducible vector based RNAi in eukaryotic cells. This methodology has potential for dramatically enhancing the ability to study intrinsic molecular mechanisms governing SSC self-renewal and differentiation. However, vector-based RNAi technologies have not been evaluated in SSCs. Thus, it is essential to examine the efficacy of inducible RNA to reduce specific gene expression in SSCs. In this proposal the mouse SSC culture system and functional transplantation assay will be utilized to achieve this objective. The three proposed specific aims are: 1) Examine the efficacy of vector based RNAi for silencing specific gene expression in SSCs, 2) Evaluate the Tet-On system for inducible RNAi in SSCs, and 3) Examine the efficacy of inducible RNAi to silence genes essential for SSC self-renewal. Development of inducible RNAi capabilities in SSCs will add an essential tool for researchers to examine the role of specific genes in SSC functions and define their modes of action. The information gained from utilization of this methodology will add essential knowledge to the field of SSC biology. These advances will have potential for correcting male infertility in humans and enhancing fertility in economically valuable and endangered species. Also, increased knowledge of intrinsic mechanisms regulating SSC function will add to the understanding of general stem cell biology and may be applicable to other adult stem cell populations. PUBLIC HEALTH RELEVANCE: Proposed experiments of this project will lead to establishment of a novel tool for studying mechanisms regulating stem cell functions in the male gonad. Knowledge gained from use of this methodology will dramatically enhance the understanding of male fertility and stem cell biology. These advances will have potential for correcting male infertility in humans and enhancing fertility in economically valuable and endangered species.